.

Speaker- Dr Bristi Chakraborty Moderator – Professor Dr Abhijit

Dutta
 THE HUMAN GENOME

● The human genome has approximately 25,000 genes that

encode the wide variety of proteins found in the human
body.
● Genes are organized into long segments of DNA, which,
during cell division, are compacted into intricate struc- tures
together with proteins to form chromosomes.
● Each somatic cell has 46 chromosomes: 22 pairs of
autosomes,, and 1 pair of sex chromosomes (XY in a male, XX
in a female).
● 1-2% coding region called exons
● 98% non coding region called introns
 MITOCHONDRIAL DNA
● Circular double stranded DNA molecule.
● 16569 base pairs- 13 protein coding regions,2
r RNA,
● 22tRNA
● Mitochondria contains 2-10 copies of
● mitochondrial dna
● Each somatic cell can have upto 1000
mitochondria
 Genetic disorders
.

MENDELIAN NON MENDELIAN

● Autosomal dominant ● Mitochondrial

● Autosomal recessive ● Triple repeat disease
● X linked dominant ● Imprinting disorder
● X linked recessive
NUMERICAL STRUCTURAL ABNORMALITIES

● Trisomy 13,18,21 ● Deletion

● Monosomy ● Duplication
● Translocation
● Inversion
 AUTOSOMAL DOMINANT INHERITANCE

● Presence of one abnormal gene in one of the autosomes

● Vertical transmission from parent to child
● Multiple generations affected
● Affected individual has 50% chance of passing the disease in future
pregnancy
● Make- female equally affected
● Examples include-hungtintons, Marfans, achondroplasia
 AUTOSOMAL RECESSIVE INHERITANCE
● mutations in both copies of a gene.
● Horizontal transmission ,the observation of multiple affected
members of a kindred in the same generation, but no affected
family members in other generations.
● recurrence risk of 25% for parents with a previous affected child;
● males and females being equally affected
● Consanguinity between parents is common.
● Examples include- cystic fibrosis, sickle cell
● anemia , lysosomal storage disorders like Tay sacks
 X LINKED RECESSIVE INHERITANCE
Males are more commonly and more severely affected than

females.

◆Female carriers are generally unaffected, or if

affected, they are affected more mildly than males.

◆Female carriers have a 25% risk for having an affected

son, a 25% risk for a carrier daughter, and a 50% chance of

having a child that does not inherit the mutated X-linked

gene.

◆ Affected males have carrier daughters and unaffected sons

because

they pass their X chromosome to all of their daughters and their Y chromosome to all of their sons. Male-to-
male transmission excludes X-linkage but is seen with autosomal dominant and Y-linked inheritance

Examples include -. Hunter, fabry,G6PD deficiency,CGD,Menkes disease,SCID.

 X LINKED DOMINANT INHERITANCE
● More lethal in males.
● No father to son transmission
● female carriers typically manifest abnormal findings.
● An affected man will have only affected daughters and unaffected
sons, and half of the offspring of an affected woman will be
affected
● Examples include- Retts,fragile X,Alpert syndrome,
● Charcot Marie tooth disease
 MITOCHONDRIAL INHERITANCE
● mitochondrial genome is entirely derived from the mother
because sperm contain few mitochondria
● A woman with a mitochondrial genetic disorder can have affected
offspring of either sex.
● an affected father cannot pass on the disease to his offspring
● Examples of mitochondrial disorders include
● MELAS (myopathy, encephalopathy, lactic acidosis,strokelike
episodes),
● MERRF (myoclonic epilepsy associated with ragged red fibers)
● Kearns-Sayre syndrome (ophthalmoplegia,
pigmentary retinopathy,cardiomyopathy)
 IMPRINTING DISORDER

● People inherit two copies of their genes—one from their mother and one
from their father. Usually both copies of each gene are active, or
“turned on,” in cells
● . In some cases, however, only one of the two copies is normally turned
on.This phenomenon is known as genomic imprinting where the
imprinted gene is silenced

● In genes that undergo genomic imprinting, the parent of origin is often

marked, or “stamped,” on the gene during the formation of egg and
sperm cells.
● This stamping process, called methylation, is a chemical reaction that
attaches small molecules called methyl groups to certain segments of
DNA.
 UNIPARENTAL DISOMY
● Uniparental disomy (UPD) occurs when a person receives two copies
of a chromosome, or part of a chromosome, from one parent and no
copies from the other parent.

● In many cases, UPD likely has no effect on health or development.

Because most genes are not imprinted, it doesn’t matter if a person
inherits both copies from one parent instead of one copy from each
parent.
● In some cases, however, it does make a difference whether a gene is
inherited from a person’s mother or father. A person with UPD may lack
any active copies of essential genes that undergo genomic imprinting.
This loss of gene function can lead to delayed development, intellectual
disability, or other health problems.
 PRADER WILLI VS ANGELMAN SYNDROME

● A classic example of imprinting

disorder is seen in Prader-Willi
syndrome and Angelman
syndrome,associated with deletion of
the same region in the proximal long
arm of chromosome 15.
● A deletion on the paternally
derived chromosome causes Prader-
Willi syndrome, in which the
maternally derived copy is still intact
but some of the imprinted genes within
this region normally remain silent.
● In contrast, a maternal deletion of the
same region causes Angelman
syndrome, leaving intact the
paternal copy that in this case has
genes that are also normally
silent.
● Obesity ● Intellectual
● Floppies disability
● Muscle hypotonia ● Ataxia
● Disorders of sexual ● Happy
development demeanour
● Seizures
STRUCTURAL ABNORMALITIES
 CLASSIFICATION OF GENETIC TESTING

CYTOGENETIC STUDY MOLECULAR GENETIC STUDY

● Karyotyping ● PCR
● FISH ● SANGER SEQUENCING
● MLPA ● NEXT GENERATION SEQUENCING
● MICROARRAY
WHEN TO SUSPECT A CHROMOSOMAL DISORDER ?
● Phenotype is suggestive of known aneuploidy or a deletion /
duplication syndrome. Eg Downs, Edward or Patau syndrome
(trisomy 21,18 and 13).
● Unexplained global developmental delay or intellectual
disability with or without dysmorphism .
● Disorders of sexual development( DSD)
● Couples with recurrent pregnancy loss or birth of a child with
one or more structural malformations.
● Female with short stature and amenorrhea.
 KARYOTYPING

● Karyotyping gives information regarding number, size of

chromosomes, position of centromere ,presence of secondary
constriction.
● Numerical and structural (> 5MB) variations can be picked up.
● Not good at detecting microdeletions or duplications.
● Commonly used banding techniques:-
● as Q-banding using quinacrine,
● reverse banding (R-banding) using acridine orange,
● C-banding (constitutive heterochromatin) using barium
hydroxide,
 FLUORESCENT IN SITU HYBRIDISATION

● Targeted test using fluorescent probes complimentary to particular region of

chromosomes
● The labeled probe is exposed to the DNA on a microscope slide, typically metaphase or
interphase chromosomal DNA.
● When the probe pairs with its complementary DNA sequence, it can be then
visualized by fluorescence microscopy .
● Testing is based on suspicion of particular deletion or duplication eg Williams
syndrome or Digeorge
● the exact chromosomal location of each probe copy can be documented and often the
number of copies (deletions, duplications) of the DNA sequence as well.
● FISH can reliably detect deletions as small as 50-200 kb of DNA. This has allowed the
clinical characterization of a number of microdeletion syndrome
 MICROARRAY

This technique is based on the principle of nucleic acid hybridization using

fluorescently labelled probes to detect with a genome wide analysis of copy
number variants and copy neutral changes like uniparental disomy which
may be implicated in development of imprinting disorders.
CMA is indicated in the following:
• Multiple anomalies not specific to a well- delineated genetic syndrome
• Apparently nonsyndromic developmental
● Autism spectrum disorders
● Will detect ANY size imbalance in chromosomes including microdeletions
,microduplications
● May miss balanced changes.
 MOLECULAR LIGATION DEPENDENT PROBE
AMPLIFICATION
MLPA is a multiplex PCR assay that utilizes up to 40 probes, each specific for a
different DNA sequence (mainly exons of a specific gene of interest), to evaluate
the relative copy number of each DNA sequence. Each probe is composed of two
half-probes (5′ and 3′ half-probes), consisting of a target-specific sequence and a
universal primer sequence allowing the simultaneous multiplex PCR amplification
of all probes . In addition, one or both half-probes contain a stuffer sequence
allowing differentiation during electrophoresis of the length of the probe itself,
and, as a consequence, the size of the amplification product. The MLPA reaction
can be divided into five steps: (1) DNA denaturation and probes hybridization; (2)
ligation reaction; (3) PCR amplification; (4) separation of amplification products
by electrophoresis; (5) data analysis.
Method Advantages Disadvantages
MLPA Detects small rearrangements Cannot detect copy neutral loss of
Up to 40 targets heterozygosity.
High throughput May have problems with mosaicism, tumor
Low cost heterogeneity, or contamination with normal
cells.
FISH Detects balanced rearrangements Cannot detect copy neutral loss of
Detects mosaicism heterozygosity.
Detects tumor heterogeneity Cannot detect small rearrangements (e.g.,
Can quantify multiple copies deletions <100 kb or duplications >500 kb).
Limited number of targets and throughput.

Quantitative/Sq-PCR Detects small rearrangements and even point Test optimization and efficiency is a concern.
mutations Limited number of targets.
Can quantify multiple copies May have problems with mosaicism, tumor
Low cost heterogeneity, or contamination with normal
cells.
Southern blot Detects small rearrangements Cannot detect copy neutral loss of
Detects mosaicism heterozygosity.
Not quantitative.
Laborious and time consuming
Limited number of targets and throughput.
CGH array Can detect very small rearrangements Cannot detect copy neutral loss of
Can probe entire genome heterozygosity.
Low cost per data point Costly equipment and reagents
Low throughput
 DNA SEQUENCING BASED TESTS
SANGER SEQUENCING -
● This method is used when a patient is suspected of having a
variant within a specific gene but that gene may have many
different causative variants (allelic heterogeneity)
● still considered as the “gold standard” for mutation screening.
● However, sequencing very large genes may be practically
expensive and time consuming.
● It is also used to confirm the results of next generation sequencing.
● It is also prudent not to opt directly for Sanger Sequencing
when the clinical suspicion is of a disorder caused by several
genes.
 NEXT GENERATION SEQUENCING (NGS)

● Next generation sequencing (NGS)- This primarily

includes Whole Exome sequencing and Focused Exome
or clinical exome sequencing.
● Next generation sequencing can also be used as targeted
panels for a group of disorders with overlapping phenotypes
with inborn errors of metabolism.
● In focused exome or clinical exome sequencing , 4500-6000
genes with known disease causation are sequenced,eg a
symptom complex with overlapping spectrum like congenital
muscular dystrophy or myopathies.
● . This approach is more practical for disorders with a well-
defined clinical phenotype with a clear monogenic aetiology
● . In Whole exome sequencing, on the other hand, all the known
protein coding genes (̴ 20,000) are sequenced and it is the test of
choice for complex disease phenotype with a suspicion of
monogenic aetiology.
● Whole genome sequencing, screens the entire genome both
coding and non coding regions , exons and introns.
INTERPRETATION OF RESULTS
 TREATMENT OF GENETIC DISORDERS

PHYSIOLOGIC THERAPY-
● Used in treatment of inborn errors of metabolism- avoiding
phenylalanine in phenylketonuria, coenzyme supplementation in
methylmalonic acids is
● Bisphosphonate treatment in osteogenesis imperfecta to prevent
fractures
● Avoid cigarette smoking in alpha 1 anti trypsin deficiency.
● Pancreatic enzyme replacement in cystic fibrosis
ENZYME REPLACEMENT THERAPY-
● Useful in lysosomal storage disorders , gaucher , hurler,
hunter etc.

● STEM CELL OR ORGAN TRANPLANTATION

● GENE THERAPY - normal gene is introduced using
viral
● or non viral vectors
 GENETIC COUNSELLING
Providing accurate knowledge to family
regarding:-
● Knowledge and diagnosis of particular condition
● Natural history of the disease
● Genetic aspect of condition and recurrence risk
● Prenatal diagnosis and prevention
● Therapies and referral
● Support group
● Follow up
● Non directive counselling
 CLASSIFICATION OF CONGENITAL ANOMALIES
Malformation: Primary intrinsic developmental defect usually caused by genetic/
environmental/ multi-factorial causes (recurrence risk varies accordingly) which
occur during the period of organogenesis which is up to 8 weeks post fertilization for
most organs. E.g. neural tube defect, ventricular septal defect, polydactyly etc.
Deformation: Distortion of a normally developed structure caused by mechanical
forces usually in the latter half of gestation and most often involving musculo-
skeletal tissues. E.g. club foot, torticollis, plagiocephaly etc.
Disruption: Breakdown of an intrinsically normally developing/ developed tissue
due to some disruptive event such as a mechanical, vascular or infectious insult.
E.g. amniotic band sequence.
Dysplasia: Abnormal cellular organization within a tissue, almost always of genetic
origin. E.g. skeletal dysplasias.
HOLOPROSENCEPHALY
Differential Associated Potential Potential genetic
diagnosis features evaluation study

TRISOMY 13 Holoprosencephaly Head ultrasound Karyotyping

Microphthalmia/ Ophthalmologic
colobomas evaluation
Congenital heart Echocardiogram
defects Renal ultrasoun
Cutis aplasia

Trisomy 18 Prominent occiput Echocardiogram Karyotyping

Micrognathia Renal ultrasound
Congenital heart
defects
Horseshoe kidney
Overlapping fingers

Single gene Microcephaly Head MRI imaging Sequencing of

disorders Hypotelorism Dental evaluation SHH, ZIC2, SIX3,
Nasal hypoplasia in those where TGIF1, GLI2,
Midline cleft lip with teeth have erupted PTCH
or without palate
Single central
incisor
COMMON INHERITED MOVEMENT DISORDERS IN CHILDREN
Common hereditary Inheritance disorder Gene responsible Genetic testing
movement disorders

FRIEDRICH’S ATAXIA AR FXN PCR AND FRAGMENT

ANALYSIS FOR
TRINUCLEOTIDE REPEATS

ATAXIA TELANGIECTASIA AR ATM SEQUENCING/NGS

SPINOCEREBELLAR ATAXIA AD ATXN 1-3, SPTBN2 PCR, FRAGMENT ANALYSIS
FOR TRINUCLEOTIDE
REPEAT SEQUENCING

WILSON’S DISEASE AR ATP7B Sequencing/NGS

LYPOSOMAL STORAGE AR GBA SMPD 1 Sequencing/NGS
DISORDERS LIKE NPC 1 AND NPC 2
NIEMANN PICK B/C,
GAUCHER, NEURONAL
CEROID LIPOFUSCINOSIS
COMMON INHERITED NEUROMUSCULAR AND NEUROCUTANEOUS
DISORDERS
Common hereditary neuromuscular & Inheritance pattern Paediatric subpopulation Responsible genes or Genetic testing
neurocutaneous disorders (incidence) chromosome

Charcot-Marie-Tooth (1/2,500) AD, AR, X-linked Adolescence Duplication of MPLA,

chr.17p11.2, CMA,
involving PMP22, sequencing/NGS
EGR2, MPZ, etc.
Myotonic dystrophy I & II (1/8,000) AD Any age DMPK & CNBP Fragment analysis for
trinucleotide or
tetranucleotide
repeats expansion
DMD (1/3,600) X-linked Childhood (boys) DMD Sequencing, MPLA
BMD (1/17,000) X-linked Adolescent DMD Sequencing, MPLA
SMAs (1/8,000) AR • Infantile- SMN1 MPLA
X-Linked adolescence UBA1 Sequencing/NGS
X-Linked • Newborn boys ATP7A
AR • Childhood IGHMBP2
• Infantile, childhood

Neurofibromatosis (1/4,000) AD, de novo Newborn, infantile, NF1 Sequencing/NGS

childhood
Tuberous sclerosis (1/15,000) AD Infant to adolescence TSC1, Sequencing/NGS
TSC2
MIDs (12/100,000) (I) MELAS & MERRF Most are nuclear Childhood, MT-TK, Sequencing/NGS
(II) KSS DNA coded AR. Some Adolescent Childhood MT-TL1,
are mitochondria MT-TH,
inheritance MT-TS1
 POLYDACTYLY

● Polydactyly is a common congenital anomaly and can

occur on the ulnar (postaxial) or the radial (preaxial)
aspects of the extremities. Of the two types, postaxial
polydactyly is the most common.
● Postaxial polydactyly can manifest as a fully
developed digit (Type A) or as a rudimentary
cutaneous appendage (Type B). Type B polydactyly
generally occurs as an isolated autosomal dominant
condition with reduced penetrance.
● In contrast, preaxial polydactyly is less common,
with a prevalence of up to 1 in 3000 live births
but occurs more frequently in Caucasians. It also
is associated with an increased incidence of
systemic conditions like Fanconi anemia,
chromosomal abnormalities, and VACTERL
association .
● Therefore, the finding of preaxial polydactyly
DIFFERENTIAL Associated Potential POTENTIAL
DIAGNOSIS features evaluation GENETIC STUDY

FANCONI ANEMIA Microcephal Hematologic Chromosomal

y Short studies breakage
stature including studies
Pigmentary complete Molecular
abnormaliti blood count testing; at least
es Thumb and bone 16 genes,
abnormaliti marrow including
es aspirate Renal FANCA and
(absent/ ultrasound BRCA2
hypoplastic
, bifid,
duplicated, etc)
Genitourinary
VACTERL Vertebral defects
abnormalities Abdominal x- Non
Anal atresia/
Pancytopenia rays AP and e
imperforate anus lateral
Cardiac defects radiographs of
Tracheoesophage the entire spine
al fistula Echocardiogram
Limb Radiographs of
anomalies affected limbs
 VENTRAL WALL DEFECTS

DIFFERENTIAL ASSOCIATED SYNDROME POTENTIAL GENETIC

DIAGNOSIS TESTING

OMPHALOCELE ● Beckwidth wideman Syndrome specific

● CHARGE study or chromosomal
● Trisomy 13 microarray
● Trisomy 18
● VACTERL

GASTROSCHISIS None None

Omphalocele

Gastroschisis
CARDIAC DEFECTS AND ASSOCIATED GENETIC
SYNDROMES
Cardiac defect Associated Features Genetic testing
syndrome

ATRIAL SEPTAL Holt oram Upper limb Sequencing of

DEFECT malformation TBX5
Cardiac
conduction
disease
COARCTATION OF Turner’s Webbed Karyotyping
AORTA posterior neck
Broad chest with
wide-spaced
nipples
Lymphedema of
hands and feet
ATRIOVENTRICUL Downs Mongoloid Karyotyping
AR CANAL slant 5th
finger
clinodactyly
CARDIAC DEFECT SYNDROME FEATURES GENETIC STUDY

PERIPHERAL Alagille Bile duct Sequencing of

PULMONARY paucity JAG1
ARTERY Butterfly
STENOSIS vertebrae
Posterior
embryotoxon
PULMONARY Noonan Tall forehead Molecular
VALVE STENOSIS Hypertelorism testing: at least
Down-slanting 12 genes,
palpebral including PTPN11
fissures Low-set,
posteriorly
rotated ears
Excess nuchal
skin Low
posterior hairline
SUPRAVALVULAR Williams Hypercalcemia Microarray or
AORTIC STENOSIS Hypotonia deletion testing
Peripheral for 7q11.23
pulmonic
stenosis
OROFACIAL CLEFTING
 CLEFT PALATE WITHOUT CLEFT LIP

Differential diagnosis Associated features Genetic study

22q11 deletion Laterally built up Chromosomal

nose microarray or FISH for
Aplasia/hypoplasia 22q11 deletion
of thymus
Hypocalcemia
Congenital heart
defects Long fingers
and toes Renal
anomalies
CHARGE Coloboma Sequencing of
Ear CHD7
anomalies
Cardiac
defects
Choanal
atresia
Genitourinary
 Differential diagnosis Associated features Genetic study

SMITH LEMLI- OPITZ Microcephaly Sequencing DHCR7

Characteristic
facial features
Cataracts
Hypospadias
Postaxial
polydactyly 2-3
toe syndactyly
STICKLER SYNDROME Myopia Sequencing of
Catara COL2A1, COL9A1,
ct COL9A2, COL11A1,
Retinal and COL11A2
detachment
Hearing loss
Spondyloephiphys
eal dysplasia
TREACHER COLIN Lower eyelid Sequencing of
SYNDROME abnormalities Microtia TCOF1, POLR1C, and
and other external ear POLR1D
abnormalities
 ESOPHAGEAL ATRESIA AND
TRACHEOESOPHAGEAL FISTULA

● Esophageal atresia (EA) is a developmental defect of the

foregut characterized by the discontinuity of the
esophagus.
● It is frequently associated with a tracheoesophageal fistula (TEF)
and in approximately half of affected individuals, the EA/TEF
anomalies are associated with other congenital anomalies
including microcephaly, single umbilical artery and duodenal
atresia.
 DIFFERENTIAL DIAGNOSIS GENETIC STUDY

INFANT OF DIABETIC MOTHER None

DOWNS SYNDROME Karyotyping

TRISOMY 18 Karyotyping

Chromosomal breakage studies

FANCONI ANEMIA Molecular testing; at least 16 genes,
including
FANCA and BRCA2

CHARGE Sequencing of CHD7

VACTERL ASSOCIATION None

THE FLOPPY INFANT
INBORN ERRORS OF METABOLISM
WHAT IS GAS CHROMATOGRAPHY -MASS
SPECTROMETRY (GC-MS)
Principle-

● The GC/ MS urinary analysis is allows simultaneous detection

of 145 + metabolites.
● As the body rapidly excretes excess abnormal metabolites in urine,
to maintain physiologic homeostasis, the detection of compounds in
urine occurs before their increase in blood level. Hence, abnormal
metabolites appear first in urine & thus presymptomatic detection is
possible by GC/ MS.

A no. of specific & precise marker compounds are designated for

congenital metabolic abnormality or for the mild nutritional
deficiencies as indicated by the presence/ absence of urinary
metabolites.
● Urine analysis using GC/MS has significantly improved the efficacy
of neonatal screening programs, demonstrating the need for more
comprehensive screening and improved throughout with
advancement in computerized MS data handling.
● With the use of noninvasive urine sample collection on dry
matrix, easy sample transport, and precise metabolome profiling,
GC/MS has provided an efficient platform for routine and high-risk
screening of IEM.
● Disorders that can be screened include-
● Organic acidurias
● Fatty acid oxidation defects
● Carbohydrate metabolism
 MONOGENIC DISEASES
● The human genome contains an estimated total of 20,000-25,000
genes that serve as blueprints for building all of our proteins.
● In single-gene diseases, a mutation in just one of these genes is
responsible for disease.
● Single-gene diseases run in families and can be dominant or
recessive, and autosomal or sex-linked.
● Pedigree analyses of large families with many affected
members are very useful for determining the inheritance
pattern of single-gene diseases.
Earlier
sanger’s
technique
used.
Now new
generation
sequencing
used.